PROJECT SUMMARY In electro-mechanical organs, such as the gastrointestinal (GI) tract, ion channels are required to generate electrical activity that drives contractions. In turn, mechanical forces affect ion channel function and therefore electrical activity, which is termed mechano-electric feedback. Therefore, ion channel mechanosensitivity is important for normal function, and abnormalities can lead to disease. In the previous grant cycles we have shown that a mechano-sensitive voltage-gated sodium channel NaV1.5, encoded by SCN5A, is present in gastrointestinal smooth muscle cells of the human small bowel and colon. Further, SCN5A mutations are associated with irritable bowel syndrome (IBS). The contribution of NaV1.5 current density and mechanosensitivity to normal and abnormal mechano-electric feedback is not known. The central hypothesis of this proposal is that NaV1.5 mechanosensitivity and current density are critical for control of human GI smooth muscle excitability, and both are regulated and targetable. We will test the central hypothesis in 3 specific aims (SAs). We will determine in: SA1 how IBS NaV1.5 mutations affect mechanosensitivity and how changes in mechanosensitivity affect mechano-electric feedback; SA2 how NaV1.5 pore determines mechanosensitivity and mechanosensitivity block by certain drugs; SA3 how NaV1.5 is regulated by miRNAs in smooth muscle cells and the effects of NaV1.5 regulation by miRNA and drugs on GI smooth muscle cell function. The SAs are supported by extensive preliminary data. 1) 30% of IBS-associated SCN5A mutations result abnormal mechanosensitivity reducing mechano-electric feedback. 2) NaV1.5 mechanosensitivity depends on ion channel pore, and NaV1.5 mechanosensitivity blockade by drugs such as ranolazine is mechanistically separate from peak current block. 3) In GI smooth muscle from slow transit constipation patients NaV1.5 is down-regulated while a small set of miRNAs is upregulated and miRNA let-7f correlates with NaV1.5 expression, down-regulates NaV1.5 current and alters electrical slow wave activity. 4) Patients on ranolazine have delayed colon transit and rat GI transit is delayed by ranolazine. To investigate the central hypothesis we use a wide variety of cutting-edge techniques, including whole-cell and single-channel voltage- and current-clamp electrophysiology and optogenetics in combination with ultra-fast pressure delivery, CRISPR-Cas to introduce patient mutations into cells, bacterial NaV channels with designer functional domains, Western blots, IHC, delivery of miRNA mimics by lentivirus, rat organotypic cultures, and a prospective clinical trial. Successful completion of the proposed studies has both basic significance and clinical impact. As a result of the work done in the previous grant cycles and the preliminary data presented in this proposal, we will significantly advance our understanding of the molecular mechanisms of NaV1.5 channel mechanosensitivity, the contribution of NaV1.5 mechanosensitivity to mechano-electric feedback and regulation of NaV1.5 in GI smooth muscle in order to understand how to target NaV1.5 to modulate abnormal GI function.